Description
Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was confirmed using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 expression without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to separate stem and progenitor cells, RNA-seq identified unique gene signatures for the separate populations which may serve as biomarkers. Pathways enrichment in stem cells identified ribosome biogenesis and membrane estrogen-receptor signaling with NF?B signaling enriched in progenitors and these were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified cancer stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland. Overall design: Comparing RNA-seq gene profiles in label-retaining prostate stem cells and non-retaining progenitor cells